The invention relates generally to compositions and methods for measuring the pH of a sample and more particularly to fluorescent protein sensors for measuring the pH of a biological sample.
The pH within various cellular compartments is regulated to provide for the optimal activity of many cellular processes. In the secretory pathway, posttranslational processing of secretory proteins, the cleavage of prohormones, and the retrieval of escaped luminal endoplasmic reticulum proteins are all pH-dependent.
Several-techniques have been described for measuring intracellular pH. Commonly used synthetic pH indicators can be localized to the cytosol and nucleus, but not selectively in organelles other than those in the endocytotic pathway. In addition, some cells are resistant to loading with cell-permeant dyes because of physical barriers such as the cell wall in bacteria, yeast, and plants, or the thickness of a tissue preparation such as brain slices.
Several methods have been described for measuring pH in specific regions of the cell. One technique uses microinjection of fluorescent indicators enclosed in liposomes. Once inside the cell, the liposomes fuse with vesicles in the trans-Golgi, and the pH of the intracellular compartments is determined by observing the fluorescence of the indicator. This procedure can be laborious, and the fluorescence of the indicator can be diminished due to leakage of the fluorescent indicator from the Golgi, or flux of the fluorescent indicator out of the Golgi as part of the secretory traffic in the Golgi pathway. In addition, the fusion of the liposomes and components of the Golgi must take place at 37xc2x0 C.; however, this temperature facilitates leakage and flux of the fluorescent indicator from the Golgi.
A second method for measuring pH utilizes retrograde transport of fluorescein-labeled verotoxin 1B, which stains the entire Golgi complex en route to the endoplasmic reticulum. This method can be used, however, only in cells bearing the receptor globotriaosyl ceramide on the plasma membrane, and it may be limited by the residence time of the verotoxin in transit through the Golgi.
In a third method, intracellular pH has been measured using the chimeric protein CD25-TGN38, which cycles as between the trans-Golgi network and the plasma membrane. At the plasma membrane, the CD25-motif binds extra-cellular anti-CD25 antibodies conjugated with a pH-sensitive fluorophore. Measurement of fluorescence upon return of the bound complex to the Golgi can be used to measure the pH of the organelle.
The invention is based on the discovery that proteins derived from the Aequorea victoria green fluorescence protein (GFP) show reversible changes in fluorescence over physiological pH ranges.
Accordingly, in one aspect, the invention provides a method for determining the pH of a sample by contacting the sample with an indicator including a first fluorescent protein moiety whose emission intensity changes as the pH varies between 5 and 10, exciting the indicator, and the determining the intensity at a first wavelength. The emission intensity of the first fluorescent protein moiety indicates the pH of the sample.
In another aspect, the invention provides a method for determining the pH of a region of a cell by introducing into the cell a polynucleotide encoding a polypeptide including a first fluorescent protein moiety whose emission intensity changes as the pH varies between 5 and 10, culturing the cell under conditions that permit expression of the polynucleotide, and determining the intensity at a first wavelength. The emission intensity of the first fluorescent protein moiety indicates the pH of the sample.
In a further aspect, the invention provides a functional engineered fluorescent protein whose amino acid sequence is substantially identical to the amino acid sequence of the 238 amino acid Aequorea Victoria green fluorescence protein shown in FIG. 3 of U.S. Ser. No. 08/911,825, now issued U.S. Pat. No. 6,054,321 (SEQ ID NO: 2), and whose emission intensity changes as pH varies between 5 and 10.
In another aspect, the invention provides a polynucleotide encoding the functional engineered fluorescent protein.
The invention also includes a kit useful for the detection of pH in a sample, e.g., a region of a cell. The kit includes a carrier means containing one or more containers comprising a first container containing a polynucleotide encoding a polypeptide including a first fluorescent protein moiety whose emission intensity changes as the pH varies between 5 and 10.